A) Immunohistochemistry of LH-RH and S-100 protein
1) Sagittal center plane
The PP of the pituitary gland was not observed in the sagittal center (SC) plane. A few capillaries were found on and in the ŽÒbottom of the floorŽÓ of the third ventricle instead. A few anti-LH-RH reaction products were observed as fine lines and numerous dotted lines on the front half of the floor of the third ventricle. Although most dotted lines of deposits ran through to the PPVM just below the third ventricle, a few arrays of deposits of the reaction ran down to the capillaries at the ŽÒbottom of the floorŽÓ. At APVM and PPVM, a few deposits were observed as dotted lines just below the third ventricle and almost all lines of deposits showed direction to the infundibular stalk. A few reaction end products were also observed at THI and a few lines of the deposits were observed from the THI to the infundibular stalk (Fig. 3L). Blood vessels were clearly observed between the brain tissue and the pars nervosa and vessels entering brain tissue were observed (Fig. 2-1). Contrary to this, no blood vessels and no pars tuberalis areas were observed in the bend of the floor of the third ventricle.
Anti-S-100 reactions in the brain in the SC plane were observed as usual. The reaction end products were mainly observed on the glia cells and their cell processes. Whereas almost all of the processes crossed the floor and reached the bottom of the floor of the third ventricle, no processes traversed to the pituitary section such as the APV or PPV (Fig. 3S). On the other hand, only cells weakly positive for S-100 protein were observed in the PPV (Fig. 3S).

2) One hundred micrometers from the SC (100um-SC)
At 100um-SC, a few primary plexus with short PP were attached to the ŽÒbottom of the floorŽÓ (Fig. 4L). It was clearly observed that the arrays of dots of anti-LH-RH reaction products ran to the plexus at PP. Although the primary plexus already joined to portal veins, a few arrays of the dots ran toward PPV. Dots of reaction of LH-RH in the THI were increased (Fig. 4L). Though the BFT area was avascular, LH-RH positive dots were numerous and the array seemed to come from the paraventricular area (Fig. 4L). At this plane, no pars tuberalis area was observed between the infundibular stalk and the hypothalamus indicated as BFT in figure 2.
At 100um-SC, the third ventricle was narrowed dorsally. Clusters of densely S-100 positive cells were observed in the PPV area. The clusters showing the apical cell processes were clearly observed (Fig. 4S). In the anterior pituitary, near the junction between the anterior lobe and the pars tuberalis, the population of S-100 positive cells was mildly increased.

3) Two hundred micrometers from the SC (200um-SC)
At 200um-SC, PP was lengthened and numerous primary plexuses were observed under the ŽÒbottom of the floorŽÓ (Fig. 5L-1). The arrays of dots of anti LH-RH reaction products running to the plexus at PP area increased drastically in this area. It was very interesting that an area of little or no reaction was observed at APVM in this plane. It was often observed on PPVM at this plane that the ŽÒbottom of the floorŽÓ faced PPV between the portal veins and sometime touched with PPV (Fig. 5L-2). The interval between the pars tuberalis and the ŽÒbottom of the floorŽÓ was clearly observed in other portions (such as PP) (Fig. 5L-3). Blood vessels were found in the THI area and numerous LH-RH positive dots were found near the BFT (Fig. 5L-1). A few slender LH-RH positive fibers with small varicosities were observed in the pars tuberalis and then disappeared around the tuberal cells in the PPV (Figs. 5L-3 and -4). Most of the cells were S-100 positive cells by immunohistochemistry. This observation was also found in mirror sections of 100um-SC and 300um from the SC (300um-SC), but were scarce.
Anti-S-100 reactions in the brain on 200um-SC were observed as usual. Only the basal part of the third ventricle was observed. While only few S-100 positive cells existed on the APV, numerous clusters of densely S-100 positive cells were observed in the PPV area (Fig. 5S-1,2). The clusters showing the apical cell processes were also clearly observed (Fig. 5S-2). A small area of the pars tuberalis was observed within the BFT.

4) Three hundred micrometers from the SC (300um-SC)
Only the margin of the third ventricle was observed at this plane (300um-SC). The primary plexus was also not observed and only PP fragments were observed under the ŽÒbottom of the floorŽÓ (Fig. 6L). While the PP and the primary plexus were no longer visible, the LH-RH deposits were widely observed (Fig. 6L). At the PPVM, it was easy to find the array arrangement to the PPV. At this plane, the pars tuberalis was observed within the BFT (Fig. 6L).
Anti S-100 reactions in the brain 300 um-SC were observed as usual. Numerous clusters of densely S-100 positive cells were still observed in the PPV area. The cluster of cells showed fine and intertwining cell processes (Fig. 6S).

B) Immunohistochemistry of LH-RH and S-100 protein on mirror sections
It was interesting to note on the mirror sections of the anti-LH-RH and S-100 studies that small round deposits of LH-RH reaction were observed around some S-100 positive cells in the PPV 200um-SC (Figs. 7L and S). The cell bodies were also positive for LH-RH (Figs. 7L and S). A few slender LH-RH positive fibers with small varicosities were observed in the pars tuberalis and then disappeared around S-100 positive cells in the PPV (Figs. 5L-3 and –4 and 7L and 7S). This observation was also found in mirror sections of 100um- and 300 um-SC, but were scarce.

C) Electron microscopy
Following observations were most obvious at 200um-SC, bot also observed at 100um-SC and 300um-SC. At PPM, numerous nerve fibers were found on the upper side (brain side) of the capillaries. In the nerve fibers, secretory granules (80-120 nm in diameter) and small vesicles (50-80 nm in diameter) were stuffed. The other side of the capillaries faced to glandular or epithelial cells of pars tuberalis. The pituitary gland and the brain were clearly separated by basement membrane of capillaries or some fibroblastic cells in this portion. At APV and APVM, similar observations as PP were obtained. The capillaries were occasionally observed between the brain and pars tuberalis (Fig. 8-1). At APV, granulated cells contained small granules (100-150 nm in diameter) were observed between the agranulated cells (Fig. 8-1). Clusters of the agranulated cells that arranged around the follicle lumen were observed. The agranulated cells projected microvilli and cilia into the lumen (Fig. 8-1). Some of the floor of the third ventricle protruded toward PPV of the pars tuberalis (Fig. 8-1).
The nerve fiber bundle with dense granules, which diameter was 100 nm, were invaded into the pars tuberalis and contacted to the agranulated cells without the basement membrane (Fig. 8-1). At the near of the site, a small cell process contacted to an agranulated cell (Fig 8-2). The small cell process contained numerous small granules (80-110 nm in diameter) and vesicles (50-100 nm in diameter). Concerning to this diameter and density, the granules corresponded to granules in nerve fibers in the median eminence (Fig 8-1), whose granules were considered as neuroendocrine granules. Agranulated cells contained cell fragments which contained numerous dense cored granules, small vesicles and mitochondria (Fig. 8-2). The size of the fragment showed good agreement to round deposits of LH-RH reactions. Although, both cell and the fragment were clearly separated by two layers of plasma membranes of both agranulated cell and fragment, no basement membrane was observed between the cell space (Fig. 8-2). The agranulated cells arranged around the small follicle (double arrows in Figs. 8-1 and 8-2).

DISCUSSION
This study demonstrated that the neuroendocrine fibers terminated not only in the primary plexus region (PP in Fig. 2) but also within the caudal part of the pars tuberalis (PPV in Fig. 2) of the pituitary gland. In addition, immuno-histochemically LH-RH positive clusters existed around the S-100 positive cells in the pars tuberalis.
Although there are numerous reports about the localization, morphological characteristics, gene expression, and regulation of the gonadal-pituitary axis of LH-RH and LH-RH neurons (1-12, 30), only few reports have referred to the existence of LH-RH positive nerves in the pars tuberalis (2). A detail of the distribution of the LH-RH positive nerve fibers in the periventricular area including the median eminence was demonstrated in the 1970ŽÕs and 1980ŽÕs (1, 2, 3, 4, 10, 13, 14). According to Naik (2) and Foster and Youngai (10), the LH-RH positive nerve fibers mainly derived from the preoptic and prechiasmatic area, arcuate nuclei and ventrolateral-premammillary body in rats and rabbits. In the present study, the LH-RH positive nerve fibers running down to the ŽÒbottom of the floorŽÓ in the cephalad portion of the third ventricle floor (PPM) were mainly derived from the nucleus situated in the cephalad part of the hypothalamus. These fibers terminated mainly on the primary plexus. Contrarily, the fibers running down to the ŽÒbottom of the floorŽÓ in the caudal portion of the floor of the third ventricle (PPVM) were derived from the nucleus in the ventrolateral-premammillary body. The LH-RH fibers were supplied from both the cephalad and the caudal parts of the hypothalamus. In addition, there were a few LH-RH fibers to the ŽÒbottom of the floorŽÓ in the middle part indicated as APVM in figure 1.
Upon reconstruction of a frontal plane view from the total of sagittal sections, the distribution pattern of the LH-RH positive nerve fibers showed a specific pattern of localization. While the fibers were numerous in zones 200 um lateral from the SC, only a few fibers were found in the very central area of the hypothalamus. On the other hand, the PP and primary plexus were clearly observed in zones 200 um lateral from the SC, while few PP and primary plexus were observed in zones 100 um lateral from the SC. The primary plexus and pars tuberalis expanded with distance from the SC up to 200 um, then rapidly decreased. These distribution patterns of the LH-RH positive fibers and primary plexuses were reasonable with regard to the architecture of the hypothalamic nuclei in the hypothalamus, and the high density of hypophyseal arteries in the lateral sides of infundibular stem.
In the earlier stages of electron microscopic research on the rat anterior pituitary gland, Farquhar (19) reported the agranular cells arranged around a follicle to be adrenocorticotropic hormone (ACTH) secreting cells. Subsequently, chromophobic cells surrounding the follicle were named "folliculo-stellate cells" based on their morphological characteristics, and were proven to have cytoplasmic contents including S-100 protein (20) and ß-adrenergic receptors (31). Based on the existence of S-100 protein in the folliculo-stellate cells, Nakajima et al. (20) supposed that they had the neuro-ectodermal origin. It has been generally accepted that they are not adrenocorticotropic cells but a distinct cell type.
The pars tuberalis has recently gained attention for its role in the photoperiodic regulation of hormone secretion in the anterior pituitary gland; especially prolactin and gonadotropin (15-18). These are commonly recognized to be related with the melatonin receptors on the cells in the pars tuberalis.
According to Morgan et al. (15), few follicular cells were observed in the ovine pars tuberalis. In our observation on the pars tuberalis of the rat pituitary gland, S-100 positive cells were clearly seen in zones 100-300 um lateral from the center of the pars tuberalis. It has been widely accepted that S-100 is the marker protein of the folliculo-stellate cells in the rat (Nakajima et al., 1980). This could be observed only in the primary plexus region in this study.
In the present study, the existence of LH-RH reactions around the S-100 positive cells in the pars tuberalis was clearly demonstrated by immunohistochemical studies of mirror sections. It is strongly suggested that the LH-RH was incorporated into the S-100 positive cells. The phenomenon of non-synaptic release of Gn-RH was reported in the fresh fish brain from immuno-electronmicroscopy studies (30). According to these, Gn-RH was released non-synaptically from dense cored vesicle (DCV) containing fiber varicosities and it exerted its modulatory action on Gn-RH receptors located on nearby as well as distant target neurons. The present study demonstrated that the neuroendocrine fibers and their varicosities were found in the pars tuberalis and nearby agranular cells. It was strongly suggested that an intimate relationship existed between the agranular cells and the LH-RH containing neurons. The possibility of a certain photoperiodic regulation of the anterior pituitary hormones through LH-RH neurons to folliculo-stellate cells has been proposed by many investigators. We believe there is a more positive action between the LH-RH neurons and the folliculo-stellate cells in the pars-tuberalis and the hormone regulating process in the pars-distalis.
Though the gap junctional connections within the anterior pituitary gland are considered to be stable, the gap junctional connections between agranular cells in the pars tuberalis may not be so stable (32). We speculated that the "message" of LH-RH could be transmitted by the network system of interconnected folliculo-stellate cells through gap junctions.
The anterior lobe of the rat pituitary gland contains few nerve fibers (33-36). The avenues for LH-RH to reach the anterior lobe from posterior or intermediate lobes are remote (37) since there are only few direct anatomical connections (38, 39). Thus, a more rapid conduction system must be employed such as interconnected folliculo-stellate cells from the pars tuberalis to the pars distalis. Our previous studies showed that gap junctional connections existed only between folliculo-stellate cells in case of adult rats (25). No gap junctions were observed before day 20 but once they appeared, the density of gap junctions in the gland increased with development until the time the animals (Wistar-Imamichi rats) had fully matured, by approximately 45 days (27). Thereafter, gap junction numbers were influenced by the concentration of sex steroids, which fluctuates with the development of the reproductive axis, estrous cycle, pregnancy and ovariectomy/castration (40-42). The findings from our previous reports indicate that the agranular folliculo-stellate cells play an important role in the pituitary gland, perhaps by participation in the regulation of hormone secretion.
It can be estimated that each folliculo-stellate cell participates in the formation of three gap junctions. If information enters the cell from one of the three gap junctions and this information is transported to neighboring cells via the other two gap junctions, the number of folliculo-stellate cells possessing the information can be raised exponentially. When this information conduction is achieved twenty times, the total number of the cells with the information reaches about one million. One million is almost equal to the total numbers of the cells composing the whole anterior pituitary gland of a rat (25). Gap junctions are composed of transmembrane channels allowing the free transcellular exchange of small cytoplasmic molecules such as Ca2+ ions. The cell-to-cell passage of Ca2+ ion can produce Ca2+ signalling observed within pituitary glands (43, 44).
When one folliculo-stellate cell in the pars tuberalis catches the information from LH-RH, this information will be easily transmitted throughout the anterior pituitary gland via gap junctions existing between folliculo-stellate cells. Moreover the "message" is conducted to target cells such as gonadotrophs by paracrine effects from the folliculo-stellate cells (45, 46). Thus we could observe discharged gonadotropes and stuffed gonadotrophs, as well as the status of their secreting cycle.

ACKNOWLEDGEMENTS
The author gives great thanks to Professor Damon C. Herbert (The Health Science Center at San Antonio, University of Texas), Dr. Nobuyuki Shirasawa (Department of Anatomy. Wakayama Medical Collage) and Professor Tsuyoshi Soji (The First Department of Anatomy, Nagoya City University, Medical School) for their support and advice during the course of this study.

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Figure Legends
Figure 1: S-100 protein positive cells in the anterior pituitary gland. Numerous positive cells and cell fragments which were cytoplasmic processes of agranulated folliculo-stellate cells were found. The cells showed polygonal irregular in shape and projected slender cytoplasmic processes between the non-reacted granulated cells. The S-100 protein is one of maker protein of the folliculo-stellate cell of the rat or human anterior pituitary. The reaction of S-100 protein showed that the reaction processed normally.
S-100 protein reaction X100
Figure 2-1: Immunohistochemistry of LH-RH reaction in the SC plane. Reactions were observed in the brain including the cephalad part of floor of the third ventricles. No reactions were observed in the pituitary area at this magnification. The infundibular stalk was connected to the pars nervosa and the floor. Around the ependymal layer, the reactions were observed. Blood vessels entered the brain. Rectangular part was schematically represented in Figure 2-2.
Anti-LH-RH stain X 20
Figure 2-2: A schematic representation of a sagittal section of the hypothalamus and the pars tuberalis of the pituitary gland. The pars tuberalis was tentatively separated into three portions for convenience of explanation from the cephalad to the caudal part. Primary plexus portion: PP. Cephalad half of the portal vein segment: APV. Caudal half of the portal vein segment: PPV. Similarly, the floor of the third ventricle was separated into three portions. Portion of the median eminence that corresponded to the primary plexus portion of pars tuberalis: PPM; anterior portion of the median eminence that corresponded to the portal vein segment of the pars tuberalis: APVM; caudal portion of median eminence corresponded to the portal vein segment of the pars tuberalis: PPVM. A small protuberance indicated by the arrow was named the transitional zone of the hypothalamus and infundibular recess (THI). A bend of the floor that was corresponded to the THI was named bend of the floor of the third ventricle (BFT). Arrowheads indicate the area expressed as "bottom of the floor" in the text.
Next 12 photographs (figures 3 to 6) are attached capital ŽÒLŽÓ or ŽÒSŽÓ following the figure number such as Figure 3L or Figure 3S. The ŽÒLŽÓ indicates Immunohistochemistry of LH-RH and the ŽÒSŽÓ indicates Immuno-histochemistry of S-100 protein.
Figure 3L: Immunohistochemistry of LH-RH on sagittal center sections. A few capillaries were attached to the ŽÒbottom of the floorŽÓ of the third ventricle. Some reaction end products were observed as dotted lines on the front portion of the floor of the third ventricle. A few arrays of deposits ran down to the capillaries, almost all of the dotted lines ran to PPVM. At the APVM and PPVM, arrays of deposits were observed just below the third ventricle and almost all of the lines were directed to the infundibular stalk. A few reaction end products were also observed from the THI to the infundibular stalk. Blood vessels were clearly observed between the brain tissue and the pars nervosa and vessels entering brain tissue were observed. Note disappearance of the PP of the pituitary gland in the SC plane. A short primary plexus (PP) attached to the ŽÒbottom of the floorŽÓ. The arrays of dots of anti-LH-RH reaction products ran on to the plexus at the PP area. A few arrays of the dots ran toward PPV. The dots of reaction of LH-RH increased in the THI. The BFT area was avascular.
X 100
Figure 3S: Immunohistochemistry of S-100 protein on sagittal center section. Anti-S-100 reactions in the brain at the SC were observed as usual. Only weakly positive cells were observed in the PPV. Intense reaction for S-100 was observed on the folliculo-stellate cells and their cell processes in the anterior lobe. Photographs of anti-S-100 reactions of the next three sagittal planes (100, 200 and 300um-SC) in higher magnifications showed findings with more clarity.
X 100
Figure 4L: At 100um-SC plane, the third ventricle was narrowed. Indicated area was a nonreaction area in the APVM.
X 100
Figure 4S: A few S 100 positive cells were observed in the pars tuberalis.
X 100
Figure 5L-1: At 200um-SC plane, numerous primary plexus were observed in the PP, which were not observed at the SC and 100um-SC. The arrays of dots of LH-RH reaction to the plexus at the PP area increased with increment of the plexus. It was very interesting though it was often on the PPVM that the arrays of deposits of anti-LH-RH reaction products ran to pars tuberalis.
X 100
Figure 5S-1: In contrast to the APV where few S-100 positive cells were seen, clusters of strongly S-100 positive cells were observed in the PPV area. The clusters showing the apical cell processes were also clearly observed.
X 100
Figure 5L-2: At the PPVM in this plane, the ŽÒbottom of the floorŽÓ approached and sometimes touched the PPV.
X 400
Figure 5S-2: High magnification of the rectangular part in figure 5S-1. S-100 positive cells were strongly observed in the PPV area. The clusters of cells showed interwinding cell processes.
X 400
Figures 5L-3 and -4: High magnification photographs at PPV. Arrows indicated slender LH-RH positive fibers in the pars tuberalis. In this area, the fiber was often observed. Arrowheads indicated the small round positive sites around the cells. Note the disappearance of a slender LH-RH fiber and a few varicosities at the cells.
X 400
Figure 6L: At 300um-SC plane, slender margin of third ventricle was observed. Only fragments of PP without primary plexus were observed. The array arrangements of LH-RH positive deposits on the APVM were increased. On the contrary to this observation for the APVM and APV, it was easy to find the arrangement within the PPV and the PPVM.
X 100
Figure 6S: At 300um-SC plane, a margin of third ventricle was observed. Few S-100 positive cells were observed.
X 100
Figure 7: Mirror images of Anti-LH-RH (7L) and S-100 (7S) stains 200um-SC. Mirror sections of the anti-LH-RH and S-100. Several round deposits of LH-RH reaction were observed (7L) around the S-100 positive cell (7S) in the PPV. Note the disappearance of a slender LH-RH fiber and a few varicosities (7L) around an S-100 positive cell (7S).
X 1,000
Figure 8-1: Non-myelinated nerve fibers (asterisk) and fragments of neuroendocrine cell in agranulated cells in PPV. The fragments stuffed numerous dense granules (arrow) (80-110 nm in diameter). The agranulated cells arranged around the small follicle (double arrow). Fragments of neuroendocrine cell in agranulated cells in PPV. The fragments contained dense granules (arrows). The dense granules were packed by membrane. No basement membrane was observed between the cell and the fragments. PP; Protrusion of the hypothalamus, PT; Pars tuberalis, NGC; Nongranulated folliculo-stellate cell
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Figure 8-2: Fragments of neuroendocrine cell (asterisk) attached on agranulated cells at PPV. At PPV and PPVM, a small cell process contacted to an agranulated cell. The cell process contained small granules (80-110 nm in diameter). The cell process touched on the agranulated cell. The agranulated cells arranged around the small follicle (double arrow). PT: Pars tuberalis
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